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Scaleup Issues for Single Use and Steel

New approaches to scaleup and methods for overcoming issues that may impact or impair scaleup were two key subjects discussed at the recent Sartorius Stedim Biotech “European Upstream and Downstream Technology Forum.”

Topics covered included both single-use and steel production systems, but the former led to a lot of discussion based on the increasing application of single-use systems in biomanufacturing activities. For example, even though a growing number of bioprocessors are turning to single use, techniques are still being developed for successful bioproduct transfer and optimization with these instruments.

According to Dr. Gilbert, the cell optimization process was carried out using a stringent medium, which was then enriched during several feeds for the production step. The challenge was to transfer and optimize the costs and the production yield without increasing the medium and feed expenses.

“We succeeded in this effort by using the Biostat® STR single-use bioreactor,” Dr. Gilbert explained. “The bioreactor geometry, the shear stress, and the dissolved O2 and CO2 distribution of this system are suitable for the cell amplification for this process, resulting in improved cell growth compared to stainless-steel bioreactors.”

Dr. Gilbert’s team currently employs single-use bioreactors with an extensive array of production systems, including mammalian, insect, and bacterial cells for multiple types of products, such as viral vectors and recombinant proteins. These operations are commonly carried out using the 50 and 1,000 L single-use bioreactors and, for some specialty activities, single-use bags up to 3,000 L.

Going the Other Way

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Technicians at Bayer Healthcare’s Berkeley, CA facility, which is committed to the production of large-scale quantities of GMP-level recombinant antihemophilic Factor VIII.

One of the main challenges for single-use bioreactor users is scalability and transfer when you need to go from disposable systems to traditional stainless-steel cultivation technologies. This was addressed by Rüdiger Heidemann, Ph.D., senior staff development scientist, Christopher Cruz, senior associate development scientist, both of whom work at Bayer Healthcare on antibody development.

According to the scientists, an important factor in comparing single-use and multiple- use bioreactor technologies is the frequency with which production will be switched from one cell line to another. This is a relatively new area of endeavor for the company’s Berkeley, CA-based site. The facility is currently committed to producing a recombinant antihemophilic Factor VIII therapy for the treatment of hemophilia A patients.

In preparation for their move into antibody production, Cruz and his colleagues have been comparing single-use bioreactors commonly employed during early process development with reusable stainless steel and/or glass vessels. Their research program aims to improve processing using fed-batch and perfusion technologies.

“For stable molecules, such as monoclonal antibodies, we favor fed-batch technology. That is where we currently implement the single-use bioreactors,” said Dr. Heidemann. “But for our perfusion platform, we employ stainless-steel vessels as the preferred option.”

Given that the company currently has a full antibody pipeline, Dr. Heidemann is looking for a quick product changeover, obtainable with disposables.

“We really have to distinguish 1,000 L and 2,000 L production levels for clinical manufacturing,” he continued. “But with commercial demands that may go up to ton quantities, we would probably use stainless steel. We do not foresee that in the near future disposables will be able to handle large commercial volumes, unless the market demand would allow smaller batch sizes”

According to Cruz, neither approach is 100% secure. “Disposables tend to have a higher failure rate as they are subject to shipping and handling issues. If the bag fails, it sets you back, so we have to make sure it is as intact as possible,” Cruz explained. “Of course, stainless-steel vessels can fail, too, for example, due to defective or improperly installed gaskets or clamp connections, or any other operational mishaps.”

Dealing with Leachables

Gerhard Greller, Ph.D., R&D director for upstream technology at Sartorius Stedim Biotech, focused on the optimization of single-use bioreactors. With the widespread adoption of single-use systems, the problem of leachables from the materials used to construct the plastic is becoming more significant.

“If you are ordering 50 L of media at a time, you may need to store it for weeks,” explained Dr. Greller. “That means these substances can drastically affect the growth of cells.”

Dr. Greller and his team have identified many of these compounds and redesigned the film that lines the bag. “We had to start from the resin selection. Through this approach, we went through a rapid learning curve, allowing us to characterize the leachables, including materials released by the gamma-irradiation process.”

Dr. Greller mentioned the work of Matthew Hammond and his coworkers at Amgen, who have identified one of the most toxic extractable compounds from a number of substances present in the polymer films used in presterilized, single-use biomanufacturing systems.

This molecule, bDtBPP, is detrimental to the growth of a number of strains of CHO cell lines, even at concentrations as low as 100 µg/L. It is derived from the compound tris(2,4-di-tert-butylphenyl)phosphate, and affects the mitochondrial membrane potential. Ionizing radiation, used in the sterilization process, appears to be the mechanism by which bDtBPP is generated.

Another critical quality control issue emphasized by Dr. Greller is the monitoring of leakage through suitable testing programs. The test method must effectively identify potential damage to installed bioreactor bags, including the bag seals, port welds, connections, and bag surfaces, which could be damaged during their assembly. These failures could cause substantial financial losses as well as constituting a significant safety hazard.

To deal expeditiously with the need for effective leak monitoring, Dr. Geller’s colleague, Martin Dahlberg, uses the Sartocheck®4 Plus Bag tester with a bag-tester fleece that prevents masking of any leaks that may have been introduced during installation.

“It allows point-of-use leak testing of single-use bioreactor bags post-installation and pre-use in its final bag holder,” said Dr. Geller.

The goal of these advances is to ensure the end user does not have to touch the bag and accessory components any more than necessary.”

Continuous Cell Culture Processing

The broad adoption of single-use technology presents particular opportunities for protein production in continuous culture of mammalian cells, especially as companies prepare for the move to scaleup. According to Christel Fenge, Ph.D., production specialist at Sartorius Stedim Biotech, nutrients may be delivered by perfusion culture or concentrated fed-batch mode operation.

“With continuous culture we can maintain higher cell densities and higher titers in a stable environment, but the process is more complex,” Dr. Fenge stated. “As a result, this is a more demanding protocol that runs a higher risk of failure and calls for higher training requirements for personnel.”

But can single-use bioreactors measure up to the challenges of such conditions? Dr. Fenge and her coworkers have grappled with the needs of high O2 demands, sufficient CO2 removal, long operating times, and elimination of proteinaceous aerosol formations.

They determined that the best oxygen transfer was achieved with a microsparger and Rushton marine impeller combination. Effective CO2 partial pressure control was achieved using a microsparger in a 50 L program at a high cell density. Effective off-gas treatment was performed with a single-use exhaust cooler, a component of the Biostat single-use bioreactor.

According to Dr. Fenge, perfusion is the most suitable approach for labile or difficult-to-express products. It can reduce process development time, provide a more stable environment, and perform efficiently in smaller production bioreactors, and it requires fewer seed steps.

“We have found that perfusion fits well into the current biopharma trends,” Dr. Fenge said. “It adapts well to production of biosimilars, the expression of new monoclonal antibody formats, and the need for smaller production quantities.”

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